Glycosyl hydrolase with beta-xylosidase and beta-glucosidase activities and uses thereof

Abstract

A novel glycosyl hydrolase with activities of beta-xylosidase and beta-glucosidase is provided. Said glycosyl hydrolase can convert 7-xylosyltaxane compounds to 7-hydroxyltaxane compounds.

The invention claimed is:  
     
       1. A glycosyl hydrolase comprising an amino acid sequence that has at least 95% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 2, wherein said glycosyl hydrolase has glycosyl hydrolase activity and is recombinantly produced by expressing said glycosyl hydrolase in a  Pichia pastoris  host cell. 
     
     
       2. The glycosyl hydrolase of  claim 1 , wherein the glycosyl hydrolase has β-xylosidase activity. 
     
     
       3. A  Pichia pastoris  host cell comprising a recombinant expression plasmid, wherein the recombinant expression plasmid comprises a polynucleotide encoding the glycosyl hydrolase of  claim 1 . 
     
     
       4. A method for hydrolyzing a glycoside compound comprising a xylosyl group and/or a glucosyl group, the method comprising
 contacting the glycosyl hydrolase of  claim 1  with the glycoside compound to remove the xylosyl group and/or to remove the glucosyl group of the glycoside compound by hydrolysis. 
 
     
     
       5. A method for hydrolyzing glycoside compounds comprising a xylosyl group and/or a glucosyl group, the method comprising
 contacting the host cell of  claim 3  with the glycoside compounds to remove the xylosyl group and/or to remove the glucosyl group of the glycoside compounds by hydrolysis. 
 
     
     
       6. The method of  claim 5 , wherein the glycoside compounds comprising a xylosyl group are selected from taxane-xyloside compounds. 
     
     
       7. The method of  claim 6 , wherein the taxane-xyloside compounds are selected from 7-xylosyltaxane compounds. 
     
     
       8. The method of  claim 7 , wherein the 7-xylosyltaxane compounds are selected from
 7-xylosyltaxol, 
 7-xylosyl-10-deacetyltaxol, 
 7-xylosylcephalomannine, 
 7-xylosyl-10-deacetylcephalomannine, 
 7-xylosyltaxol C, 
 7-xylosyl-10-deacetyltaxol C, 
 7-xylosylbaccatin III, and 
 7-xylosyl-10-deacetylbaccatin III. 
 
     
     
       9. The method of  claim 8 , wherein the products obtained after removing the xylosyl group by hydrolysis with the glycosyl hydrolase are 7-hydroxyltaxane compounds selected from
 paclitaxel, 
 10-deacetyltaxol, 
 cephalomannine, 
 10-deacetylcephalomannine, 
 taxol C, 
 10-deacetyltaxol C, 
 baccatin III, and 
 10-deacetylbaccatin III. 
 
     
     
       10. The method of  claim 7 , wherein the 7-xylosyltaxane compounds are selected from mixtures of 7-xylosyltaxane compounds. 
     
     
       11. The method of  claim 10 , wherein the mixtures of 7-xylosyltaxane compounds are from plant tissues of the  Taxus  genus, cell cultures of plants of the  Taxus  genus, or cell cultures of microorganisms capable of generating 7-xylosyltaxane compounds. 
     
     
       12. The method of  claim 11 , wherein the plants of the  Taxus  genus are selected from  T. baccata, T. brevifolia, T. wallichiana, T. media, T. chinensis, T. yunnanensis , and  T. cuspidate.    
     
     
       13. The method of  claim 11 , wherein the plant tissues of the  Taxus  genus are selected from roots, needles, bark or whole seedlings. 
     
     
       14. The method of  claim 7  wherein a solvent is used in the hydrolysis of the 7-xylosyltaxanes, wherein the solvent is water, methanol, ethanol, ethyl acetate, acetone, n-hexane, chloroform, dichloromethane, N,N-dimethylformamide (DMF), or dimethyl sulfoxide (DMSO). 
     
     
       15. A method for hydrolyzing lignocelluloses to obtain monosaccharides, the method comprising contacting the lignocelluloses with the glycosyl hydrolase of  claim 1  together with cellulases and/or hemicellulases to hydrolyze lignocelluloses to obtain monosaccharides.